In vitro culture of high-purity osteoclasts induced by macrophage colony stimulating factor/receptor activator of nuclear factor kappa B ligand: Optimal dosage investigation / 中国组织工程研究

ABSTRACT

BACKGROUND: Macrophage colony-stimulating factor (M-CSF)/receptor activator of nudeer kappa B ligand (RANKL), two types of cytokines co-induce myeloid stem cells to form osteoclasts, is a kind of new method to harvest ostaoclasts with high purity and quantity, but there is lack of uniform cultivation standard. OBJECTIVE: To construct an effective M-CSF/RANKL induced mice myeloid stem cells inducing osteoclast differentiation cultivation system. METHODS: Myeloid stem cells ware obtained from ICR mice and then cultured for 24 hours in a-minimum essential medium containing M-CSF, at cell density of 10~7/L, 10~8/L, 10~9/L. Then 10 μg/L M-CSF and 20, 50, 100 μg/L RANKL were added into culture medium. Tartaric-resistant acid phosphatase stained was performed to observe the transition process from stem cell to osteoclast, as well as cell morphology and stain situation after culture, and positive stained osteoclasts were counted. We compared the influence of different induction conditions to the quantity of osteoclast. RESULTS AND CONCLUSION: A small quantity of osteoclasts contained many red positive beads in the intracytoplasm were observed at 3 days. There were positive beads with hypochromatic dikeryon in cells. A large amount of positively stained osteoclests were seen after 6-day cultudng, which maintained dikaryon. After 9-day culturing, positively stained colossal multinudear cells occurred, became larger and maintained three nuclei. At certain cell density, 100 μg/L RANKL could induce to form more osteoclasts compared with other 2 concentrations (P < 0.05); at certain RANKL concentration, the osteoclasts formation at cells density of 10~8/L was dramatically greater than other 2 cell densities (P < 0.05); the number of osteoclasts was the most when the concentration of RANKL was 100 μg/L and cell density of 10~8/L (P < 0.05). When osteoclasts are induced by M-CSF/RANKL from mudne myeloid stem cells, the best concentration of RANKL is 100 μg/L and cells density is 10~8/L.